Manish Gupta1, Pengfei Song, Charles R Yates, Bernd Meibohm. 1. Department of Pharmaceutical Sciences, College of Pharmacy, The University of Tennessee Health Science Center, 874 Union Avenue, Suite 5p, Memphis, TN 38163, USA.
Abstract
BACKGROUND: The human chemokine receptor CXCR2 (IL8RB) is a high affinity receptor for interleukin-8 as well as other CXC chemokines, and is involved in the chemotaxis of immune cells. Genetic variants of CXCR2 have potential relevance in various inflammatory human disorders. We developed a real-time polymerase chain reaction (PCR)-based allelic discrimination assay for the detection of the CXCR2 single nucleotide polymorphisms (SNPs) C785T, T1208C and G1440A. METHODS: Polymorphisms were delineated using PCR amplification of specific alleles (PASA). Allele-specific primers were developed for both wild-type and mutant alleles. An additional nucleotide mismatch at the third position from the 3' end of each primer was used to improve amplification specificity and to prevent generation of nonspecific products. Genotypes were assigned based on PCR growth curves and melt curve analysis performed on a SmartCycler using SYBR Green I chemistry. RESULTS: Genotyping assignments were successfully performed in a set of 20 human DNA samples, and were validated by comparison with results from direct DNA sequencing and agarose gel electrophoresis of PCR products. CONCLUSIONS: Due to its rapid and relatively inexpensive performance and accuracy, the presented allelic discrimination assay for CXCR2 polymorphisms has wide applicability, especially for high-throughput sample analysis in large population genotyping studies.
BACKGROUND: The human chemokine receptor CXCR2 (IL8RB) is a high affinity receptor for interleukin-8 as well as other CXC chemokines, and is involved in the chemotaxis of immune cells. Genetic variants of CXCR2 have potential relevance in various inflammatory human disorders. We developed a real-time polymerase chain reaction (PCR)-based allelic discrimination assay for the detection of the CXCR2 single nucleotide polymorphisms (SNPs) C785T, T1208C and G1440A. METHODS: Polymorphisms were delineated using PCR amplification of specific alleles (PASA). Allele-specific primers were developed for both wild-type and mutant alleles. An additional nucleotide mismatch at the third position from the 3' end of each primer was used to improve amplification specificity and to prevent generation of nonspecific products. Genotypes were assigned based on PCR growth curves and melt curve analysis performed on a SmartCycler using SYBR Green I chemistry. RESULTS: Genotyping assignments were successfully performed in a set of 20 human DNA samples, and were validated by comparison with results from direct DNA sequencing and agarose gel electrophoresis of PCR products. CONCLUSIONS: Due to its rapid and relatively inexpensive performance and accuracy, the presented allelic discrimination assay for CXCR2 polymorphisms has wide applicability, especially for high-throughput sample analysis in large population genotyping studies.